The mechanism of specific binding of free cholesterol by the steroidogenic acute regulatory protein: evidence for a role of the C-terminal α-helix in the gating of the binding site

Abstract

Steroidogenesis depends on the delivery of free cholesterol to the inner mitochondrial membrane by StAR (steroidogenic acute regulatory protein). Mutations in the StAR gene leads to proteins with limited cholesterol-binding capacity. This gives rise to the accumulation of cytoplasmic cholesterol, a deficit in steroid hormone production and to the medical condition of lipoid congenital adrenal hyperplasia. A detailed understanding of the mechanism of the specific binding of free cholesterol by StAR would be a critical asset in understanding the molecular origin of this disease. Previous studies have led to the proposal that the C-terminal alpha-helix 4 of StAR was undergoing a folding/unfolding transition. This transition is thought to gate the cholesterol-binding site. Moreover, a conserved salt bridge (Glu169-Arg188) in the cholesterol-binding site is also proposed to be critical to the binding process. Interestingly, some of the documented clinical mutations occur at this salt bridge (E169G, E169K and R188C) and in the C-terminal alpha-helix 4 (L275P). In the present study, using rationalized mutagenesis, activity assays, CD, thermodynamic studies and molecular modelling, we characterized the alpha-helix 4 mutations L271N and L275P, as well as the salt bridge double mutant E169M/R188M. The results provide experimental validation for the gating mechanism of the cholesterol-binding site by the C-terminal alpha-helix and the importance of the salt bridge in the binding mechanism. Altogether, our results offer a molecular framework for understanding the impact of clinical mutations on the reduction of the binding affinity of StAR for free cholesterol.

The Novel Mutation p.Trp147Arg of the Steroidogenic Acute Regulatory Protein Causes Classic Lipoid Congenital Adrenal Hyperplasia with Adrenal Insufficiency and 46,XY Disorder of Sex Development

“…This hydrophobic cluster around this residue has been proposed to stabilize the C-terminal helix in a closed conformation [30]. It has been suggested that the α4 helix would undergo unfolding during ligand binding [30,31]. The conservation of Trp147 across the domain family indicates that a lipid-binding mechanism via local unfolding or a significant conformational change in the C-terminal helix could be a family-wide phenomenon.…”

“…4). This hydrophobic cluster around this residue has been proposed to stabilize the C-terminal helix in a closed conformation [30]. It has been suggested that the α4 helix would undergo unfolding during ligand binding [30,31].…”

Validation of the mechanism of cholesterol binding by StAR using short molecular dynamics simulations

“…These side chains create hydrophobic anchoring points favorable for a stabilizing clamp between ␣-helix 4 and the core of the protein. It is expected that upon binding of cholesterol to StAR, this clamping mechanism would help stabilize the complex by shielding it from the solvent exposure, until its later release during the steroidogenesis [22]. To gain insight into the molecular behavior of StAR at the atomic level, we conducted MD simulations on the WT protein, the ␣-helix 4 mutants (F267Q, L271N, and L275P) and the salt bridge (E169M/R188M) mutant and emphasized on the variation of their radius of gyration (Rg).…”

“…We hypothesize that these clinical mutations alter the binding affinity of StAR for cholesterol and that this promotes its accumulation in LCAH [22]. Alteration of the ligand binding affinity of a protein requires an understanding of the effect of mutating individual residues in the ligand-binding site and the overall structure of the protein [28].…”

“…First, the cholesterol binding site, a hydrophobic cavity with a putative salt bridge between Glu 169 and Arg 188 , is responsible for ligand specificity and orientation within the cavity. Second, the C-terminal ␣-helix 4 stabilizes the holocomplex and gates the transfer of cholesterol [22].…”